Unformatted text preview: Restric(on Enzymes aka __________________ What are they/What do they do? • Enzymes that recognize/bind and cleave speciﬁc 4‐8 bp sequences in dsDNA, called “restric'on sites” • crea(ng reproducible restric'on fragments” • Most restric(on sites are short inverted sequences (palindromes!) Usually dimers that recognize both strands Bacteria Destroy foreign DNA methyla(on 1. S(cky ends w/5’ overhang 2. S(cky ends w/3’ overhang 3. Blunt (ﬂush) ends (DISADVANTAGE = less eﬃcient liga(on) AluI, BamHI, EcoRI, HindIII Structure Naturally occurring in what organism? Na(ve func(on/purpose? How does bacteria protect its own DNA? Ways they cut (3) Common ones Prac(ce Problems 1. Restric(on enzyme X recognizes a 5bp sequence, how frequently will it cut? about every ___45___ bps 2. Construct a restric(on map of a linear fragment of DNA, using the following data. Your map should indicate the rela(ve posi(ons of the restric(on sites along with distances from the ends of the molecule to the restric(on sites and between restric(on sites: DNA Sizes of Fragments (bp) uncut DNA 10,000 DNA cut w/ EcoRI 8000, 2000 DNA cut w/ BamHI 5000, 5000 DNA cut w/EcoRI + BamHI 5000, 3000, 2000 On a linear axis, from 0 to 10kb (10,000bps) – 2 possible answers (lem or right column below) Scenario 1 Scenario 2 BamHI cuts @ 5kb, EcoRi @2kb BamHI cuts @ 5kb, EcoRI cuts @8kb 0 to 2kb 2kb fragment 0 to 5kb 5kb fragment 2 to 5kb 3kb fragment 5 to 8kb 3kb fragment 5 to 10kb 5kb fragment 8 to 10kb 2kb fragment 3. Shown below is a ﬁc((ous 4,000bp plasmid. Create a ﬁgure predic(ng the appearance of a gel showing digests with EcoRI, BamHI, and EcoRI + BamHI combined. Include a lane showing a ladder for size reference. Ladder sizes will vary, but should correspond to indicated band sizes • EcoRI only : single 4,000bp band • BamHI only: three bands, ~ 2000bp, ~1600bp, ~300bp • EcoRI+BamHI: 4 bands, ~2000bp, ~1000bp, ~600bp, ~300bp A new plasmid DNA has been isolated from E.coli. It was found to have a single site for the restric(on enzyme SmaI. Explain how you can use electron microscopy to determine whether the plasmid replicates unidirec(onally or bidirec(onally. Do a restric(on digest with SmaI to linearize the plasmid DNA Observe with electron microscopy Unidirec(onal replica(on: replica(on bubble should only be expanding in one direc(on rela(ve to the cut site Bidirec(onal replicia(on: replica(on bubble should expand in both direc(ons from the SmaI cut site Bacteria Genome Plasmid Circular, dsDNA Eukaryotes Linear, dsDNA (many) λ phage outside host Inside host Linear, dsDNA Circular, dsDNA shape+make‐up Circular, dsDNA Mode of replica(on #replicons #forks/replicon Bi‐direc(onal mustache 1 2 unidirec(onal 1 1 Bi‐direc(onal mustache many 2 Rolling circle Name and BRIEFLY describe (3 sentences MAX) 4 diﬀerent proteins involved in the pre‐inita'on of DNA replica(on at an E.coli Ori (PRIOR to DNA polymerase III). (a) topoisomerase: relieves strain on dsDNA that results from strand separa(on by nicking and allowing free rota(on of DNA (Type I makes single stranded breaks, Type II makes double stranded breaks) (b) helicase: ATP dependent enzyme that melts dsDNA (separates by breaking hydrogen bonds) (c) Single stranded binding proteins (SSBPs): bind to separated DNA strands and keep them from reassocia(ng (Addi(onal minor func(ons: protect ssDNA, s(mulate helicase ac(vity) (d) DNA primase: synthesizes short RNA primers 10‐12bps long for DNA pol III to extend from In the Meselson & Stahl experiment E.coli cells were transferred from 15N containing heavy medium into 14N containing light medium and samples were removed at various (mes for analysis of the DNA by CsCl equilibrium centrifuga(on. What frac'on of the DNA would be expected at light, hybrid and heavy densi(es a?er 3 doublings based on the following models of replica(on: (A) Semi‐conserva've Light ____3/4_____ Hybrid ____1/4____ Heavy ____none____ (B) Conserva've Light ____7/8_____ Hybrid ____none____ Heavy _____1/8___ ...
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- Spring '08
- DNA, dsDNA, new plasmid DNA, foreign DNA methyla, plasmid DNA Observe, ) uncut DNA